The goal of the proposed research is to complement, extend and enrich our research funded under CA669800 "Biological Activity of HPV E7 in Human Epithelial Cells." We will use raft cultures to determine the relative importance of certain biochemical and biological activities of E7 to induce centrosome abnormalities, aberrant mitoses and aneuploidy in basal keratinocytes. We also propose to generate "mixed-culture rafts" where normal and HPV-16 episome expressing cells are mixed at various ratios and grown on rafts. Individual cells will be identifiable by expression of a fluorescent marker. This will allow us to model early HPV-associated dysplastic lesions where a small number of HPV-infected cells grow and expand surrounded by a large number of normal cells. We will perform histological analyses and evaluate expression of marker proteins such as p63 and p16INK4A to determine whether dysplasia and/or expression of such markers is confined to HPV expressing cells. These experiments will also reveal whether there are functional interactions between adjoining normal and HPV expressing cells. In addition we will determine the contribution of the spatial orientation of the mitotic spindle to the architecture of a squamous epithelium and whether centrosome abnormalities lead to a disruption of this spacial organizing function, potentially contributing to dyplasia.